N2-fixation, ammonium release and N-transfer to the microbial and classical food web within a plankton community

Abstract

We investigated the role of N2-fixation by the colony-forming cyanobacterium, Aphanizomenon spp., for the plankton community and N-budget of the N-limited Baltic Sea during summer by using stable isotope tracers combined with novel secondary ion mass spectrometry, conventional mass spectrometry and nutrient analysis. When incubated with (15)N2, Aphanizomenon spp. showed a strong (15)N-enrichment implying substantial (15)N2-fixation. Intriguingly, Aphanizomenon did not assimilate tracers of (15)NH4(+) from the surrounding water. These findings are in line with model calculations that confirmed a negligible N-source by diffusion-limited NH4(+) fluxes to Aphanizomenon colonies at low bulk concentrations (<250 nm) as compared with N2-fixation within colonies. No N2-fixation was detected in autotrophic microorganisms <5 μm, which relied on NH4(+) uptake from the surrounding water. Aphanizomenon released about 50% of its newly fixed N2 as NH4(+). However, NH4(+) did not accumulate in the water but was transferred to heterotrophic and autotrophic microorganisms as well as to diatoms (Chaetoceros sp.) and copepods with a turnover time of ~5 h. We provide direct quantitative evidence that colony-forming Aphanizomenon releases about half of its recently fixed N2 as NH4(+), which is transferred to the prokaryotic and eukaryotic plankton forming the basis of the food web in the plankton community. Transfer of newly fixed nitrogen to diatoms and copepods furthermore implies a fast export to shallow sediments via fast-sinking fecal pellets and aggregates. Hence, N2-fixing colony-forming cyanobacteria can have profound impact on ecosystem productivity and biogeochemical processes at shorter time scales (hours to days) than previously thought.

Figure 1

Bulk sample enriched with Aphanizomenon sp. and incubated with ¹³C-labeled bicarbonate and ¹⁵N₂. Isotope ratios of ¹⁵N/¹⁴N versus ¹³C/¹²C in various organisms (a) incubated from 0300 to 1500 hours and (b) incubated from 0300 to 0300 hours: Aphanizomenon sp. vegetative cells (green symbols); autotrophic bacteria attached to Aphanizomenon sp. (yellow symbols); free-living autotrophic bacteria (red symbols); heterotrophic bacteria attached to Aphanizomenon sp. (cyan symbols); free-living heterotrophic bacteria (black symbols); and diatoms (violet symbols). The dotted lines represent the isotope ratios of the natural abundance of ¹⁵N/¹⁴N and ¹³C/¹²C.

Figure 2

Bulk (<5-μm fraction) sample incubated with ¹³C-labeled bicarbonate and ¹⁵N₂. Isotope ratios of ¹⁵N/¹⁴N versus ¹³C/¹²C in cells incubated from 2100 to 0900 hours (open symbols) and incubated from 0900 to 2100 hours (closed symbols). The dotted lines represent the natural abundance levels of ¹³C/¹²C and ¹⁵N/¹⁴N isotope ratios.

Figure 3

Ratio of ¹⁵NH₄⁺ tracked in the bulk water to ¹⁵N₂-fixation during the 24-h experiment. The symbols show the average value with the s.d. (n=3) of the average value shown as bars.

Figure 4

Bulk sample enriched with Aphanizomenon sp. incubated with ¹³C-labeled bicarbonate and ¹⁵NH₄⁺ during 5 h in light: isotope ratios of ¹⁵N/¹⁴N versus ¹³C/¹²C in various organisms: Aphanizomenon sp. vegetative cells (green symbols); autotrophic bacteria attached to Aphanizomenon sp. (yellow symbols); free-living autotrophic bacteria (red symbols); heterotrophic bacteria attached to Aphanizomenon sp. (cyan symbols); free-living heterotrophic bacteria (black symbols); and eukaryotes (violet symbols). The dotted lines represent the isotope ratios of the natural abundance of ¹⁵N/¹⁴N and ¹³C/¹²C.

Figure 5

Total NH₄⁺ (¹⁵NH₄⁺ and ¹⁴NH₄⁺) (closed symbols) in the bulk and the added tracer ¹⁵NH₄⁺ (open symbols) measured as a function of time in light.

Figure 6

The theoretical shortest time required to cover N-demand for one cell division at diffusion limitation at a NH₄⁺ bulk concentration of 250 nm (for details, please see text).